It is well established that there is a dynamic relationship between the expanding tumor and the host surrounding tissue. Cancer-associated fibroblasts (CAFs), the most common cellular population found in the tumor microenvironment, supporting tumor growth and dissemination. Here, we set out to determine the factors that may be involved in dramatic alteration of gene expression pattern in CAFs, focusing on microRNA and transcriptional regulators. We established matched pairs of human CAFs isolated from endometrial cancer and normal endometrial fibroblasts. MicroRNA and mRNA analyses identified differential expression of 11 microRNAs, with miR-31 being the most downregulated microRNA in CAFs (p = 0.007). We examined several putative miR-31 target genes identified by microarray analysis and demonstrated that miR-31 directly targets the homeobox gene SATB2, which is responsible for chromatin remodeling and regulation of gene expression, and was significantly elevated in CAFs. The functional relevance of miR-31 and SATB2 were tested in in vitro models of endometrial cancer. Overexpression of miR-31 significantly impaired the ability of CAFs to stimulate tumor cell migration and invasion, without affecting tumor cell proliferation. Genetic manipulation of SATB2 levels in normal fibroblasts or CAFs showed that, reciprocally to miR-31, SATB2 increased tumor cell migration and invasion, while knockdown of endogenous SATB2 in CAFs reversed this phenotype. Introduction of SATB2 into normal fibroblasts stimulated expression of a number of genes involved in cell invasion, migration and scattering. These findings provide new insights into tumor-stroma interaction and document that miR-31 and its target gene SATB2, are involved in regulation of tumor cell motility.
The tumor microenvironment has an important role in cancer progression. Here we show that miR-148a is downregulated in 15 out of 16 samples (94%) of cancer-associated fibroblasts (CAFs) compared with matched normal tissue fibroblasts (NFs) established from patients with endometrial cancer. Laser-capture microdissection of stromal cells from normal tissue and endometrial cancer confirmed this observation. Treatment of cells with 5-aza-deoxycytidine stimulated the expression of miR-148a in the majority of CAFs implicating DNA methylation in the regulation of miR-148a expression. Investigation of miR-148a function in fibroblasts demonstrated that conditioned media (CM) from CAFs overexpressing miR-148a significantly impaired the migration of five endometrial cancer cell lines without affecting their growth rates in co-culture experiments. Among predicted miR-148a target genes are two WNT family members, WNT1 and WNT10B. Activation of the WNT/β-catenin pathway in CAFs was confirmed by microarray analysis of gene expression and increased activity of the SuperTOPFIash luciferase reporter. We found elevated levels of WNT10B protein in CAFs and its level decreased when miR-148a was re-introduced by lentiviral infection. The 3′-UTR of WNT10B, cloned downstream of luciferase cDNA, suppressed luciferase activity when co-expressed with miR-148a indicating that WNT10B is a direct target of miR-148a. In contrast to the effect of miR-148a, WNT10B stimulated migration of endometrial cancer cell lines. Our findings have defined a molecular mechanism in the tumor microenvironment that is a novel target for cancer therapy.
Cancer growth and metastasis involve an active interaction between primary tumor cells and the tumor's microenvironment, which consists of fibroblasts, vascular cells and inflammatory cells. Such interactions between the cancer and the cells within the microenvironment induce differential expression of genes that act to foster the growth, invasion and spread of tumor cells. This study utilizes pair-wise primary cultures of fibroblasts from normal and cancerous human endometrial tissues of the same patients to investigate the potential factors in the tumor microenvironment and their role in carcinogenesis. Both microRNA and mRNA profiling were performed on paired normal and cancer fibroblasts. SatB2 emerged as a promising candidate when its expression was shown to be significantly increased in fibroblasts surrounding the tumor tissues in all but one patients tested (n=8). Further investigation documented that SatB2 is a target for miR31, via 2 binding sites located within the SATB2 3’ UTR region. In concordance, miR31 is down-regulated in tumor fibroblasts, which further implicates the potential role of SatB2 in promoting a favorable environment for tumors. Ectopic expression of SATB2 in normal fibroblasts does not affect the growth rate of an endometrial cancer cell line (EC-1) in co-culture experiments; however, it did increase the motility of EC-1 cells towards these fibroblasts by multiple folds in both in vitro migration and invasion assays. A reduction of SATB2 expression in cancer activated fibroblasts (CAFs) by shRNA attenuated the migratory ability of EC-1 cells, further supporting the role of SatB2 in tumor aggression. Our observations also indicated that the conditioned media from CAFs were sufficient to induce the motility changes in the EC-1 cells. We believe the elucidation of SatB2 pathway(s) will assist in uncovering the intricate connections between tumors and their microenvironments, leading to new targets for treating tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1423.
The dynamic interactions between tumor cells and their corresponding microenvironment play an important role in carcinogenesis. Fibroblasts are a dominant feature within a tumor stroma and have undergone a “reprogramming” to become cancer-activated fibroblasts (CAFs). In our study we established primary cell cultures of fibroblasts from matched pairs of normal human endometrium and endometrial cancer tissues and analyzed the differential expressions of mRNA and microRNA. We found that the WNT pathway was activated in CAFs and confirmed these results via Topflash luciferase reporter assays. Wingless-type (WNT) proteins are a highly conserved family of secreted glycoproteins that have been implicated in the carcinogenesis of many cancers. We demonstrated that one of the reasons for this WNT pathway activation was the methylation-dependent silencing of the secreted frizzled-related protein 4 (SFRP4), an antagonist to the WNT signaling pathway. An additional reason for WNT pathway activation was found in the microRNA analysis. These experiments showed downregulation of miR-148a, which is a predicted regulator of the founder member of the WNT family, WNT1. We observed that CAFs have increased WNT1 protein expression in three of the five endometrial cancer CAFs. We confirmed that miR-148a directly targets WNT1 by cloning the WNT1 3'UTR into a luciferase reporter construct and showed that co-transfection with miR-148a results in a decrease of luciferase activity. When mutations to the miR-148a binding sites were introduced into the same luciferase reporter construct, the downregulation of luciferase activity by miR-148a was prevented. We conclude that the observed activation of the WNT pathway in endometrial cancer CAFs may be regulated by the miR-148a and contribute to the progression of the endometrial cancer tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1430.
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